1. Field of the Invention
This invention relates to immunoassaying. Immunoassayings are proving of immense value in medicine and biology for the assaying of the constituents of biological fluids, because of the sensitivity and specificity of such assaying. In immunoassaying procedures, for a given target compound, a synthetic antigen is generally first prepared. Heretofore, this has usually been accomplished by coupling the target compound, through a coupling group to a carrier which confers antigenicity to the entire compound. The compound coupled to the carrier is usually known as a hapten and, when coupled, it exhibits antigenic determinacy by causing the antibodies produced to be specific to it. Thus, the antibodies produced have a distinct and unique character, such that they will bind with only a specific compound or class of compounds. The objective in devising the synthetic antigen-hapten conjugate is to provide a compound which will generate antibodies that are specific to the target compound.
Antibodies are prepared by injecting the synthetic hapten-antigen conjugate into competent vertebrate animals and recovering blood serum from the animals after they have had time to generate antibodies. Typical and preferred animals are mammals, e.g., rabbits and goats.
The principal problem is usually synthesizing antigens that are sufficiently specific. Biological fluids such as blood and urine frequently contain very closely related compounds and it is common for antibodies to be unable to distinguish the target compound from close relatives, or sometimes even distant ones. The antibody is then considered to be a poor one and is said to have low specificity and high cross-reactivity.
The assay itself is commonly a competitive binding assay. In such an assay, the target compound, which is not necessarily extracted, is allowed to compete with known quantities of a radiation labeled standard to bind with a known quantity of a specific antibody. From measurement of the proportion of the labeling in the standard-antibody complex that results, the amount of target compound present can be calculated. Radioactive labeling is particularly convenient. Fluorescent perturbation can be used. Normally it will be necessary to remove any unreacted labeled standard, before making the determination on the antibody complex.
2. The Prior Art
The cross-referenced applications disclosed, inter alia, hapten-antigen conjugates in which a hapten is coupled to an antigen through a diazo group, a phenyl ring, and an amide bond to a proteinic antigen. Numerous haptens are disclosed, and steroids are of particular interest.
A synthesis is described in which the hapten is coupled to the amine of an aminoaryloic acid by diazotization, and the product is then coupled to an antigenic protein with an amide bond.
Other proposals for the preparation of phenethylamine conjugates have involved coupling through a functional group, such as the amine, which is undesirable.
Spector in U.S. Pat. No. 3,704,282 discloses the preparation of catecholamine antigens by carbodiimide condensation of the amine with the carboxyl group of an immunogenic material, such as a protein or polypeptide. Included within the generic definition of catecholamine are hydroxyphenethylamines, and these include norepinephrine, the latter being one of the materials of specific interest according to the present application. The antigens reported in Spector are said to be useful for raising antibodies, and radiation-labeled competition assays are described. Apparently, according to the Spector disclosure, antibody binding with norepinephrine was obtained, and there is a report of an in vitro study of its characteristics. However, Spector gives no cross-reactivity data, and there is no evidence indicating that the antibody generated according to Spector would be useful in assays as there is nothing to indicate the specificity or the binding affinity of the antibody.
In U.S. Pat. Nos. 2,372,066 and 2,301,532, Fell, a number of methods are disclosed for coupling of histamine, an organic ring compound with a primary amine group, to a protein for the purpose of preparing an antigenic conjugate. The objective of these patents is to produce an in vivo response, and there appears to be no consideration of the use of the antibodies in vitro. Many reaction sequences are shown, though most involve the coupling of the protein through the primary amino group. Though there are a few examples which involve coupling through the histidyl ring, the preferred route is that of Examples 1 through 4 of U.S. Pat. No. 2,372,066, the materials produced by that route reportedly producing an antigen in which the histamine is coupled to the protein through an amide bond derived, in part, from the histamine amino group, through a phenyl ring which, in turn, is coupled through a diazo group to the protein. However, histamine is not a phenethylamine, and neither of these patents suggests the coupling of phenethylamine through the ring for use in production of antigens, for subsequent production of antibodies from these antigens, and the use of the resulting antibodies with low cross-reactivity in assays for target compounds.
In "Peptides and Amino Acids," Kopple (W. A. Benjamin, Inc., 1966), pages 36 through 38, the use of blocking groups in peptide synthesis is disclosed. The use of trifluoroacetyl as an amine blocking group is specifically mentioned. However, there is no consideration of antigen synthesis or of its problems, and the teaching regarding the stability of the trifluoroacetamide suggests this to be an unsuitable blocking group in at least some of the reactions of particular interest here.